Resveratrol inhibits LPS-induced mice mastitis through attenuating the MAPK and NF-κB signaling pathway

Accepted Manuscript Resveratrol inhibits LPS-induced mice mastitis through attenuating the MAPK and NF-κB signaling pathway Xu Zhang, Yanan Wang, Chong Xiao, Zhengkai Wei, Jingjing Wang, Zhengtao Yang, Yunhe Fu PII:

S0882-4010(17)30245-0

DOI:

10.1016/j.micpath.2017.04.002

Reference:

YMPAT 2201

To appear in:

Microbial Pathogenesis

Received Date: 10 March 2017 Revised Date:

1 April 2017

Accepted Date: 3 April 2017

Please cite this article as: Zhang X, Wang Y, Xiao C, Wei Z, Wang J, Yang Z, Fu Y, Resveratrol inhibits LPS-induced mice mastitis through attenuating the MAPK and NF-κB signaling pathway, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2017.04.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Resveratrol inhibits LPS-induced mice mastitis through attenuating the MAPK and NF-κB signaling pathway Xu Zhang1, Yanan Wang1, Chong Xiao1, Zhengkai Wei1, Jingjing Wang1, Zhengtao Yang1, YunheFu1, 2* 1.

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Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Jilin University, Changchun, Jilin Province 130062, People’s Republic of China. Department of Pathogenobiology, The Key Laboratory of Zoonosis, Chinese Ministry of Education, College of Basic Medicine, Jilin University, Changchun, Jilin Province 130062, People’s Republic of China.

* Corresponding authors College of Veterinary Medicine, Jilin University, Changchun 130062, PR China E-mail address:[email protected]. Tel.: +86 431 87835140. Fax: +86 431 87835140.

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ABSTRACT Resveratrol is a natural polyphenol extracted from mangy plants. It has been reported that resveratrol show multitudinous positive role in biology such as anti-oxidant, anti-nociception and anti-inflammatory effects. Therefore, the present study devotes to test the effect of resveratrol on LPS-induced mastitis in mice. Resveratrol was administered intraperitoneally 1h before LPS treatment. And the anti-inflammatory effect of resveratrol was measured by histopathological examination, MPO assay, real-time PCR and western blotting analysis. The results showed that resveratrol significantly reduced the LPS-induced mammary histopathological changes. Meanwhile, it sharply attenuated the activity of MPO. The result also indicated that the resveratrol can decrease the expression of pro-inflammatory cytokines TNF-α and IL-1β. From the results of western blotting, resveratrol suppressed the expression of phosphorylation of p65 and IκB from NF-κB signal pathway and phosphorylation of p38 and ERK from MAPK signal pathway. These findings suggested that resveratrol may inhibit the inflammatory response in the mastitis. Keyword: Mastitis; Resveratrol; LPS; MAPK pathway

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1. INTRODUCTION Bovine mastitis is defined as inflammatory response in the mammary gland, which is a serious problem in dairy cattle and has leading to enormous financial loss around the world [1]. The mastitis can decrease the quality of milk such as the increased number of somatic cells [2]. Many kinds of factors can induce the bovine mastitis, which the most general factor is the infection of pathogens [3, 4]. For example, the Escherichia coli, which belongs to Gram-negative bacteria genera, can promote inflammation reaction in the mammary epithelial cells [5, 6]. Then, the mammary epithelial cells will produce inflammatory cytokines which have the function of promoting inflammation. At present, the major method to treat the mastitis was using antibiotic, but the abuse of antibiotic will cause some hidden troubles such as production of drug resistant, drug residue and the generation of superbugs [7, 8]. Therefore, we need carry on some additional research to find new methods which is advantageous to treat and prevent bovine mastitis. Lipopolysaccharide (LPS), the main composition of the cytoderm of gram-negative bacteria, has been known that it is generally used to establish an animal model of bovine mastitis [9, 10]. It was always used to establish the mastitis model and study the different aspects of the mastitis, such as the treatment and prevention of some highly active pharmaceutical ingredients [11]. LPS can activate many kings of signaling pathways. Toll-like receptor-4 (TLR4) is one of the main signaling pathways [12]. The activation of the TLR4 can promote the activation of nuclear

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transcription factor-kappaB (NF-κB) and the mitogen-activated protein kinase (MAPK) signaling pathway, which can promote the production of inflammatory cytokines, such as TNF-α and IL-1β [13, 14]. These cytokines are the most familiar indicators to determine the occurrence of mastitis. Resveratrol (3,4,5-trihydroxylstilbene) is a natural polyphenol present in a variety of plant species, such as peanuts and grape [15]. Resveratrol has been reported to express various kinds of activities such as anti-inflammatory and anti-oxidative effects [16, 17]. It has been reported that resveratrol expressed the treatment and prevention effects in inflammatory response of a variety of tissue damage like hepatic injury, renal injury and enteritis [18-20]. Resveratrol has been reported to inhibit LPS-induced acute lung injury in mice [21]. Also, resveratrol also inhibited LPS-induced inflammatory cytokines production in RAW264.7 cells [22]. However, there are few literature reports about the positive role of resveratrol on LPS-induced mastitis in mice. Therefore, the objective of this study is to investigate whether resveratrol has anti-inflammatory function and mechanism in LPS-induced mastitis in mice. 2. MATERIALS AND METHODS 2.1. Animals BALB/c mice, weighing from 20-25g, were provided by the Center of Experimental Animals of Bethune Medical College of Jilin University (Jilin, China). All mice get enough fresh water and fodder.7 days before experiment, mice were fed under the environment of 12/12h light/dark cycle. The temperature was maintained at 24±1℃and relative humidity was maintained at 50%~60%. All the animal experiments were finished in accordance with the guide for the Care and Use of Laboratory Animals established by the US National Institutes of Health. 2.2. Reagents LPS was purchased from the Sigma Chemical Co. (L-2880, St. Louis, MO, USA). Resveratrol (purity>99.8%) was purchased from the Pharmaceutical and Biological Products (Beijing, China). The tissue protein extraction reagent was supplied by the Thermo. The MPO determination kit was purchased from the Jiancheng Bioengineering Institute of Nanjing (Nanjing, Jiangsu Province, China). All the antibodies in western blot were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Other chemicals were conformed from reagent grade. 2.3. Experimental model and group Sixty lactational mice were randomly divided into 5 groups: Control group, LPS group, LPS + resveratrol (10, 20 or 40mg/kg) groups. The experimental mice were separated from their pups 3h before induction of LPS. The mice was hocussed and the aim area was sterilized with 70% ethanol. Their teat ducts of the fourth milk area were injected 50ml LPS (0.2mg/ml, dissolved in sterile PBS). Resveratrol was given by intraperitoneal injection 1h before LPS injection. The control group and LPS group were intraperitoneally injected with an equal volume of normal saline (NS). 24h after LPS injected, CO2 inhalation was used to kill the mice. We chose 24 h after LPS treatment was based on previous study [23]. The middle part of mammary glands were collected and stored at -80℃ for later experiment. 2.4. Histopathological examination A fraction of fresh mammary glands of each sample was excised and immobilized in 10% formalin when we collected the mammary glands. Then tissues were made into paraffin sections after dehydrated by graded alcohols and embedded in paraffin. Later, the tissue slices were stained with hematoxylin and eosin. Finally, the histopathologic changes were observed under a light microscope and collected images.

ACCEPTED MANUSCRIPT 2.5. Myeloperoxidase (MPO) assay The mammary tissue samples were homogenized and collected the liquid supernatant. Then, the MPO activity in mammary tissues were measured using detection kit according to manufacturer’s instructions. The enzymatic activity was tested at 460 nm.

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2.6. Real-time PCR The TRizol (Invitrogen, Carlsbad, CA, USA) was used to extract the total RNA. The RNA samples were dissolved into 0.1% diethyl pyrocarbonate solution (DEPC). Then, The RNA samples were reversed transcription to cDNA following the manufacturer's instructions of the Revert Aid First Strand cDNA Synthesis Kit (MBI Fermentas, Lithuania). Real-time PCR (RT-PCR) was completed on a 7500 real-time PCR system (Applied Biosystems, Carlsbad, CA, USA). Primers were acquired from Sangon Biotech Co. Ltd (Shanghai, China). The reaction conditions of PCR are as follows: the samples were incubated 2 min at 50°C, then incubated at 95 °C for 10 min; the third step was 40 cycles of 95 °C for 15 s and 60 °C for 1 min; finally, the samples were incubated at 70℃ for 10min. Pimer sequences and the reaction system were listed at table1. 2.7. Western blot analysis We weight 0.1g mammary tissues and added 0.9ml mammalian protein extraction reagent, then homogenated and centrifuged under 12000rpm for 10min at 4℃. We collected the supernatant and determine the protein concentrations by BCA protein assay kit. Every lane was added different protein with equal amounts. Meanwhile, they were separated through 10%SDS polyacrylamide gels and PVDF membranes. Then, the membranes were blocked in 3% Bull Serum Albumin (BSA) for 3h and incubated with the primary antibodies (1:1000 dilutions with TBST )at 4 °C overnight. Subsequently, the membranes were washed 3 times for 5min with Tris-buffered saline with 0.1% Tween-20 (TBST) and incubated with the secondary antibodies with 2h. Finally, the membranes were washed 3 times for 5min again and visualized with the enhanced chemiluminescence kit. 2.8. Statistical analysis All data are expressed as mean ± SEM. The differences of various experimental groups were analyzed by using one-way ANOVA combined with Tukey’s multiple comparison tests. Values of p< 0.05 were considered to be statistically significant. 3. RESULTS 3.1. Effect of resveratrol on histopathological changes As shown in Fig. 1, in the control group, the mammary tissues have no histopathologic changes (Fig. 2 A). In the LPS group, the mammary alveolus obviously got thick, edema, and a mass of inflammatory cells infiltrate into the tissue (Fig. 2 B). However, the pretreatment of resveratrol could significantly reduce the histologic changes which were caused by LPS (Fig. 2 CE). 3.2. Resveratrol attenuates MPO activity The MPO activity was a typical marker of inflammatory cell infiltration. The results showed that the MPO activity obviously increased through injecting LPS. However, the MPO activity of resveratrol prevention groups were significantly reduced in dose-dependent manner compared with LPS group (Fig.3). 3.3. Effect of resveratrol on inflammatory cytokines production

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In this study, we detected the mRNA levels of inflammatory cytokine to prove the anti-inflammatory effects of resveratrol on LPS-induced bovine mastitis. As shown in Fig.4, the mRNA levels of TNF-α and IL-1β in the LPS group were significantly higher than that in the control group. However, compared with LPS group, the mRNA levels of TNF-α and IL-1β in the LPS + resveratrol (10,20,40mg/kg) group were observably suppressed in a dose-dependent manner. The results showed that the inflammatory response was significantly inhibited by resveratrol through decreasing the inflammatory cytokines production. 3.4. Resveratrol inhibits MAPK signaling pathway activation The MAPK pathway has the function to regulate the production of inflammatory response. So we investigated the expression of p38 and ERK phosphorylation by using western blotting. The results showed that LPS significantly increased p38 and ERK phosphorylation compared with the control group. However, the pretreatment of resveratrol decreased the expression of p38 and ERK phosphorylation compared with the LPS group (Fig. 5). 3.5. Effect of resveratrol on NF-κB signaling pathway activation The NF-κB signaling pathway is also concerned with the inflammatory response in mastitis. So we measured the levels of phosphorylation of p65 and IκBα protein. The western blot results showed that the expression of p-p65 and IκBα in the LPS group was observably increased compared with that of control group. However, resveratrol attenuated the levels of p-p65 and IκBα. Above all, the resveratrol may express anti-inflammatory effect by inhibiting NF-κB signaling pathway activation (Fig.6). 4. DISCUSSION Bovine mastitis is an extremely prevalent disease in dairy cattle. The characteristic of mastitis primarily were as follows: infiltration of inflammatory cell, red and swollen of mammary gland, even damage of mammary alveolar [24-26]. In the present study, we establish the LPS-induced mice model to study the protective effects and mechanism of resveratrol. The results of histopathological examination showed that resveratrol significantly reduced the LPS-induced histopathology changes. MPO activity, a marker of neutrophil infiltration, is directly used to show the severity of inflammatory response [27]. In this study, MPO activity was significantly elevated in the LPS group and inhibited in the resveratrol groups. This phenomenon conform to the previous studies that resveratrol can express anti-inflammatory effect [28, 29]. It also indicated that resveratrol had protective effect on LPS-induced mastitis. The productions of inflammatory cytokines such as TNF-α and IL-1β can influence the inflammatory responses in the bovine mastitis [30]. TNF-α is the relatively early inflammatory medium and can promote the process of producing other inflammatory factors [31]. IL-1β, a subtype of IL-1, is released by macrophages, endothelial cell and monocytes [32]. IL-1β shows the functions that promote inflammatory response and induce the production of secondary cytokines [33]. A number of previous studies held the view that inhibiting the expressions of TNF-α and IL-1β have positive effect on LPS-induced bovine mastitis [23, 34], our results were consistent with it. In the present study, the concentrations of TNF-α and IL-1β in the LPS group were significantly higher than those in the control group. This data indicated that LPS can induce the production of TNF-α and IL-1β. However, the precaution of resveratrol attenuated the levels of TNF-α and IL-1β in a dose-dependent manner in our study. Therefore, the results indicated that

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the anti-inflammatory effects of resveratrol were possibly related to suppressing the release of the pro-inflammatory cytokines such as TNF-α and IL-1β. A number of studies showed that the NF-κB and MAPK signaling pathways could modulate the expression of TNF-α and IL-1β [35, 36]. Meanwhile, some studies indicated that the inhibition of LPS-induced inflammation in mammary gland might be completed by suppressing the activation of NF-κB signaling pathway [37]. To further investigate the anti-inflammatory mechanism of resveratrol, we detect the levels of p-p65and p-IκBα in the NF-κB signaling pathway. The data showed that the phosphorylation of p65 and IκBα protein of the LPS group was higher than the control group. However, the precaution of resveratrol attenuated the phosphorylation of p65 and IκBα in a dose-dependent manner. Therefore, the result demonstrated that the resveratrol have preventive effect against LPS-induced mastitis by inhibiting the activation of NF-κB signaling pathway. Moreover, we also measure the activation of p38 and ERK in the MAPK signal pathway. The results of phosphorylation of p38 and ERK protein showed the similar trend with phosphorylation of p65 and IκBα. All above, we confirmed that the resveratrol inhibited the activation of NF-κB and MAPK signal pathways so as to attenuate the inflammatory response of LPS-induced mice mastitis and express the protective effect. In conclusion, the present study indicated that the protective effect of resveratrol on LPS-induced mice mastitis may be related to the process of inhibiting inflammatory cytokines production, which was especially via inhibiting the activation of the NF-κB and MAPK signaling pathway. The findings suggest that resveratrol may be a potential preventive medicine of mastitis. More researches and clinical practices are needed before the resveratrol was applied in production. CONFLICT OF INTEREST The authors declare that they have no conflict of interests. ACKNOWLEDGMENTS This study was supported by grants from the National Natural Science Foundation of China (No. 31602122, 31572583) and China Postdoctoral Science Foundation funded project (2016M600233).

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ACCEPTED MANUSCRIPT [37] Li D, Chen J, Ye J, Zhai X, Song J, Jiang C, et al. Anti-inflammatory effect of the six compounds isolated from Nauclea officinalis Pierrc ex Pitard, and molecular mechanism of strictosamide via suppressing the NF-kappaB and MAPK signaling pathway in LPS-induced RAW 264.7 macrophages. Journal of ethnopharmacology. 2017;196:66-74.

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Figure legend Fig.1 The chemical structure of resveratrol. Fig.2 The effect of resveratrol on mammary tissue histopathological changes in LPS-induced mice mastitis (×400) . Mammary tissue of control group (A), the LPS group (B), the LPS + resveratrol 10 mg/kg group(C), the LPS + resveratrol 20 mg/kg group (D), the LPS + resveratrol 40 mg/kg group (E). Fig.3 Effects of resveratrol on MPO activity. The MPO activity was determined at 24 h after LPS administration. The values presented are the means ± SEM of three independent experiments. Number sign (#) indicates that the MPO activity of LPS group is significantly different from the control group(p<0.01); double asterisk(**) indicate the MPO activity is significantly different from the LPS group(p<0.01). Fig.4 The effect of resveratrol on inflammatory cytokines production. The mice were pretreated with resveratrol (10, 20, 40mg/kg) 1h before injecting LPS. The mRNA levels of TNF-α and IL-1β were measured by RT-PCR. The mRNA levels of TNF-α and IL-1β were significantly increased in the LPS group, however the pretreatment of resveratrol reduced mRNA levels of TNF-α and IL-1β in a dose-dependent manner. Fig.5 The effect of resveratrol on MAPK pathway. The values presented are the means ± SEM of three independent experiments. Number sign “#” (p<0.01) is significantly different from the control group; Number sign “**” (p<0.01) is significantly different from the LPS group. Fig.6 The effect of resveratrol on NF-κB pathway. The values presented are the means ± SEM of three independent experiments. Number sign “#” (p<0.01) is significantly different from the control group; Number sign “**” (p<0.01) is significantly different from the LPS group. Table.1 the primers which was used in the real-time PCR of this study

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TNF-α forward TNF-α reverse IL-1β forward IL-1β reverse GAPDH forward GAPDH reverse

ACGGGCTTTACCTCATCTACTC GCTCTTGATGGCAGACAGG AGGTGGTGTCGGTCATCGT GCTCTCTGTCCTGGAGTTTGC TCAACGGGAAGCTCACTGG CCCCAGCATCGAAGGTAGA

Product size 140 b p 195 b p

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Nucleotide sequence （5’-3’）

237 b p

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ACCEPTED MANUSCRIPT Highlights The model of mice mastitis was established by injection of LPS.




Resveratrol can inhibit the mRNA levels of TNF-α and IL-1β which are the main pro-inflammatory cytokines. Resveratrol can suppress the NF-κB and MAPK signal pathways in LPS-induced mice mastitis.

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